Covalent modification of a protein with the 76-residue protein ubiquitin (Ub), thereby changing the stability, localization, or activity of the target protein, regulates almost all aspects of eukaryotic cellular function. This reaction requires the sequential actions of three types of enzymes; an activating enzyme (E1) forms a thioester linkage between its catalytic cysteine and the carboxyl group of Gly76 in ubiquitin to activate it; a conjugating enzyme (E2) that receives ubiquitin from the E1; a ubiquitin ligase (E3) that transfers the ubiquitin molecule from the E2 to a substrate. For every eukaryotic species, there are two E1 enzymes, whereas approximately dozens of E2 enzymes (Table 1) and thousands of E3 ligases are found. The E3 ligases can be classified into two major catagories, the HECT domain (homologous to E6-associated protein C-Terminus)-containing ligases and those carrying a catalytic RING (really interesting new gene) domain.[unreadable] We have established an in vitro ubiquitination system using purified gp78c, a cytosolic domain of an ER-associated RING ligase and ube2g2. The assay allows us to assemble Lys48 linked polyubiquitin chains. Using this system, we have demonstrated that polyubiquitin chains can be preassembled on the catalytic cysteine of Ube2g2 before being transferred to a substrate.